Cotton cultivar 99X35

ABSTRACT

A novel cotton cultivar, designated 99×35, is disclosed. The invention relates to the seeds of cotton cultivar 99×35, to the plants of cotton 99×35 and to methods for producing a cotton plant produced by crossing the cultivar 99×35 with itself or another cotton variety. The invention further relates to hybrid cotton seeds and plants produced by crossing the cultivar 99×35 with another cotton cultivar.

BACKGROUND OF THE INVENTION

[0001] The present invention relates to a cotton (Gossypium) seed, acotton plant, a cotton variety and a cotton hybrid. This inventionfurther relates to a method for producing cotton seed and plants.

[0002] There are numerous steps in the development of any novel,desirable plant germplasm. Plant breeding begins with the analysis anddefinition of problems and weaknesses of the current germplasm, theestablishment of program goals, and the definition of specific breedingobjectives. The next step is selection of germplasm that possess thetraits to meet the program goals. The goal is to combine in a singlevariety an improved combination of desirable traits from the parentalgermplasm. In cotton, the important traits include higher fiber (lint)yield, earlier maturity, improved fiber quality, resistance to diseasesand insects, resistance to drought and heat, and improved agronomictraits.

[0003] Pureline cultivars of cotton are commonly bred by hybridizationof two or more parents followed by selection. The complexity ofinheritance, the breeding objectives and the available resourcesinfluence the breeding method. Pedigree breeding, recurrent selectionbreeding and backcross breeding are breeding methods commonly used inself pollinated crops such as cotton. These methods refer to the mannerin which breeding pools or populations are made in order to combinedesirable traits from two or more cultivars or various broad-basedsources. The procedures commonly used for selection of desirableindividuals or populations of individuals are called mass selection,plant-to-row selection and single seed descent or modified single seeddescent. One, or a combination of these selection methods, can be usedin the development of a cultivar from a breeding population.

[0004] Pedigree breeding is primarily used to combine favorable genesinto a totally new cultivar that is different in many traits than eitherparent used in the original cross. It is commonly used for theimprovement of self-pollinating crops. Two parents which possessfavorable, complementary traits are crossed to produce an F₁ (filialgeneration 1). An F₂ population is produced by selfing F₁ plants.Selection of desirable individual plants may begin as early as the F₂generation wherein maximum gene segregation occurs. Individual plantselection can occur for one or more generations. Successively, seed fromeach selected plant can be planted in individual, identified rows orhills, known as progeny rows or progeny hills, to evaluate the line andto increase the seed quantity, or, to further select individual plants.Once a progeny row or progeny hill is selected as having desirabletraits it becomes what is known as a breeding line that is specificallyidentifiable from other breeding lines that were derived from the sameoriginal population. At an advanced generation (i.e., F₅ or higher) seedof individual lines are evaluated in replicated testing. At an advancedstage the best lines or a mixture of phenotypically similar lines fromthe same original cross are tested for potential release as newcultivars.

[0005] Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., Allard, 1960; Simmonds, 1979; Sneep, et al. 1979; Fehr,1987).

[0006] The single seed descent procedure in the strict sense refers toplanting a segregating population, harvesting one seed from every plant,and combining these seeds into a bulk which is planted the nextgeneration. When the population has been advanced to the desired levelof inbreeding, the plants from which lines are derived will each traceto different F₂ individuals. Primary advantages of the seed descentprocedures are to delay selection until a high level of homozygosity(e.g., lack of gene segregation) is achieved in individual plants, andto move through these early generations quickly, usually through usingwinter nurseries.

[0007] The modified single seed descent procedures involve harvestingmultiple seed (i.e., a single lock or a simple boll) from each plant ina population and combining them to form a bulk. Part of the bulk is usedto plant the next generation and part is put in reserve. This procedurehas been used to save labor at harvest and to maintain adequate seedquantities of the population.

[0008] Selection for desirable traits can occur at any segregatinggeneration (F₂ and above). Selection pressure is exerted on a populationby growing the population in an environment where the desired trait ismaximally expressed and the individuals or lines possessing the traitcan be identified. For instance, selection can occur for diseaseresistance when the plants or lines are grown in natural orartificially-induced disease environments, and the breeder selects onlythose individuals having little or no disease and are thus assumed to beresistant.

[0009] Promising advanced breeding lines are thoroughly tested andcompared to popular cultivars in environments representative of thecommercial target area(s) for three or more years. The best lines havingsuperiority over the popular cultivars are candidates to become newcommercial cultivars. Those lines still deficient in a few traits arediscarded or utilized as parents to produce new populations for furtherselection.

[0010] These processes, which lead to the final step of marketing anddistribution, usually take from seven to twelve years from the time thefirst cross is made. Therefore, development of new cultivars is atime-consuming process that requires precise forward planning, efficientuse of resources, and a minimum of changes in direction.

[0011] A most difficult task is the identification of individuals thatare genetically superior because, for most traits the true genotypicvalue is masked by other confounding plant traits or environmentalfactors. One method of identifying a superior plant is to observe itsperformance relative to other experimental lines and widely grownstandard cultivars. For many traits a single observation isinconclusive, and replicated observations over time and space arerequired to provide a good estimate of a line's genetic worth.

[0012] The goal of a commercial cotton breeding program is to developnew, unique and superior cotton cultivars. The breeder initially selectsand crosses two or more parental lines, followed by generationadvancement and selection, thus producing many new genetic combinations.The breeder can theoretically generate billions of different geneticcombinations via this procedure. The breeder has no direct control overwhich genetic combinations will arise in the limited population sizewhich is grown. Therefore, two breeders will never develop the same linehaving the same traits.

[0013] Each year, the plant breeder selects the germplasm to advance tothe next generation. This germplasm is grown under unique and differentgeographical, climatic and soil conditions, and further selections arethen made, during and at the end of the growing season. The lines whichare developed are unpredictable. This unpredictability is because thebreeder's selection occurs in unique environments, with no control atthe DNA level (using conventional breeding procedures), and withmillions of different possible genetic combinations being generated. Abreeder of ordinary skill in the art cannot predict the final resultinglines he develops, except possibly in a very gross and general fashion.The same breeder cannot produce, with any reasonable likelihood, thesame cultivar twice by using the exact same original parents and thesame selection techniques. This unpredictability results in theexpenditure of large amounts of research moneys to develop superior newcotton cultivars.

[0014] Proper testing should detect any major faults and establish thelevel of superiority or improvement over current cultivars. In additionto showing superior performance, there must be a demand for a newcultivar that is compatible with industry standards or which creates anew market. The introduction of a new cultivar will incur additionalcosts to the seed producer, and the grower, processor and consumer; forspecial advertising and marketing and commercial production practices,and new product utilization. The testing preceding the release of a newcultivar should take into consideration research and development costsas well as technical superiority of the final cultivar. Forseed-propagated cultivars, it must be feasible to produce seed easilyand economically.

[0015] Cotton, Gossypium hirsutum, is an important and valuable fieldcrop. Thus, a continuing goal of plant breeders is to develop stable,high yielding cotton cultivars that are agronomically sound. The reasonsfor this goal are obviously to maximize the amount and quality of thefiber produced on the land used and to supply fiber, oil and food foranimals and humans. To accomplish this goal, the cotton breeder mustselect and develop plants that have the traits that result in superiorcultivars.

[0016] The development of new cotton cultivars requires the evaluationand selection of parents and the crossing of these parents. The lack ofpredictable success of a given cross requires that a breeder, in anygiven year, make several crosses with the same or different breedingobjectives.

[0017] The cotton flower is monecious in that the male and femalestructures are in the same flower. The crossed or hybrid seed isproduced by manual crosses between selected parents. Floral buds of theparent that is to be the female are emasculated prior to the opening ofthe flower by manual removal of the male anthers. At flowering, thepollen from flowers of the parent plants designated as male, aremanually placed on the stigma of the previous emasculated flower. Seeddeveloped from the cross is known as first generation (F₁) hybrid seed.Planting of this seed produces F₁ hybrid plants of which half theirgenetic component is from the female parent and half from the maleparent. Segregation of genes begins at meiosis thus producing secondgeneration (F₂) seed. Assuming multiple genetic differences between theoriginal parents, each F₂seed has a unique combination of genes.

SUMMARY OF THE INVENTION

[0018] The present invention relates to a cotton seed, a cotton plant, acotton variety and a method for producing a cotton plant.

[0019] The present invention further relates to a method of producingcotton seeds and plants by crossing a plant of the instant inventionwith another cotton plant.

[0020] This invention further relates to the seeds of cotton variety99X35, to the plants of cotton variety 99×35 and to methods forproducing a cotton plant produced by crossing the cotton 99×35 withitself or another cotton line. Thus, any such methods using the cottonvariety 99×35 are part of this invention, including selfing,backcrosses, hybrid production, crosses to populations, and the like.

[0021] In another aspect, the present invention provides for singletrait converted plants of 99×35. The single transferred trait maypreferably be a dominant or recessive allele. Preferably, the singletransferred trait will confer such traits as herbicide resistance,insect resistance, resistance for bacterial, fungal, or viral disease,male fertility, male sterility, enhanced fiber quality, and industrialusage. The single trait may be a naturally occurring cotton gene or atransgene introduced through genetic engineering techniques.

[0022] In another aspect, the present invention provides regenerablecells for use in tissue culture of cotton plant 99×35. The tissueculture will preferably be capable of regenerating plants having thephysiological and morphological characteristics of the foregoing cottonplant, and of regenerating plants having substantially the same genotypeas the foregoing cotton plant. Preferably, the regenerable cells in suchtissue cultures will be embryos, protoplasts, meristematic cells,callus, pollen, leaves, anthers, roots, root tips, flowers, seeds, orstems. Still further, the present invention provides cotton plantsregenerated from the tissue cultures of the invention.

DEFINITIONS

[0023] In the description and tables which follow, a number of terms areused. In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

[0024] Lint Yield. As used herein, the term “lint yield” is defined asthe measure of the quantity of fiber produced on a given unit of land.Presented below in pounds per acre.

[0025] Lint Percent. As used herein, the term “lint percent” is definedas the lint (fiber) fraction of seed cotton (lint and seed).

[0026] Gin Turnout. As used herein, the term “gin turnout” is defined asa fraction of lint in a machine harvested sample of seed cotton (lint,seed, and trash).

[0027] Fiber Length (Len). As used herein, the term “fiber length” isdefined as 2.5% span length in inches of fiber as measured by HighVolume Instrumentation (HVI).

[0028] Uniformity Ratio (UR). As used herein, the term “uniformityratio” is defined as a measure of the relative length uniformity of abundle of fibers as measured by HVI.

[0029] Micronaire (Mic). As used herein, the term “micronaire” isdefined as a measure of the fineness of the fiber. Within a cottoncultivar, micronaire is also a measure of maturity. Micronairedifferences are governed by changes in perimeter or in cell wallthickness, or by changes in both. Within a variety, cotton perimeter isfairly constant and maturity will cause a change in micronaire.Consequently, micronaire has a high correlation with maturity within avariety of cotton. Maturity is the degree of development of cell wallthickness. Micronaire may not have a good correlation with maturitybetween varieties of cotton having different fiber perimeter. Micronairevalues range from about 2.0 to 6.0: Below 2.9 Very fine Possible smallperimeter but mature (good fiber), or large perimeter but immature (badfiber). 2.9 to 3.7 Fine Various degrees of maturity and/or perimeter.3.8 to 4.6 Average Average degree of maturity and/or perimeter. 4.7 to5.5 Coarse Usually fully developed (mature), but larger perimeter. 5.6+Very coarse Fully developed, large-perimeter fiber.

[0030] Fiber Strength (T1). As used herein, the term “fiber strength” isdefined as the force required to break a bundle of fibers as measured ingrams per millitex on the HVI.

[0031] Fiber Elongation (E1). As used herein, the term “fiberelongation” is defined as the measure of elasticity of a bundle offibers as measured by HVI.

[0032] Plant Height. As used herein, the term “plant height” is definedas the average height in inches or centimeters of a group of plants.

[0033] Stringout Rating (So). As used herein, the term “stringoutrating” is defined as a visual rating prior to harvest of the relativelooseness of the seed cotton held in the boll structure on the plant.

[0034] Maturity Rating (Matur). As used herein, the term “maturityrating” is defined as a visual rating near harvest on the amount ofopened bolls on the plant.

[0035] Vegetative Nodes. As used herein, the term “vegetative nodes” isdefined as the number of nodes from the cotyledonary node to the firstfruiting branch on the main stem of the plant.

[0036] Seedweight (Sdwt). As used herein, the term “seedweight” is theweight of 100 seeds in grams.

[0037] Fallout (Fo). As used herein, the term “fallout” refers to therating of how much cotton has fallen on the ground at harvest.

[0038] Lint Index. As used herein, the term “lint index” refers to theweight of lint per seed in milligrams.

[0039] Seed/boll. As used herein, the term “seed/boll” refers to thenumber of seeds per boll.

[0040] Seedcotton/boll. As used herein, the term “seedcotton/boll”refers to the weight of seedcotton per boll.

[0041] Lint/boll. As used herein, the term “lint/boll” is the weight oflint per boll.

[0042] Fruiting Nodes. As used herein, the term “fruiting nodes” isdefined as the number of nodes on the main stem from which arisebranches which bear fruit or bolls.

[0043] Essentially all the physiological and morphologicalcharacteristics. A plant having essentially all the physiological andmorphological characteristics means a plant having the physiological andmorphological characteristics, except for the characteristics derivedfrom the converted trait.

[0044] Single trait Converted (Conversion). Single trait converted(conversion) plant refers to plants which are developed by a plantbreeding technique called backcrossing or via genetic engineeringwherein essentially all of the desired morphological and physiologicalcharacteristics of a variety are recovered in addition to the singletrait transferred into the variety via the backcrossing technique or viagenetic engineering.

[0045] Disease Resistance. As used herein, the term “disease resistance”is defined as the ability of plants to restrict the activities of aspecified pest, such as an insect, fungus, virus, or bacterial.

[0046] Disease Tolerance. As used herein, the term “disease tolerance”is defined as the ability of plants to endure a specified pest (such asan insect, fungus, virus or bacteria) or an adverse environmentalcondition and still perform and produce in spite of this disorder.

[0047] VRDP. As used herein, the term “VRDP” is defined as the alleledesignation for the single dominant allele of the present inventionwhich confers virus resistance. VRDP designates “Virus ResistanceDeltapine”. TABLE 1 VARIETY DESCRIPTION INFORMATION Species: Gossypiumhirsutum L. General: Plant Habit: Intermediate Foliage: IntermediateStem Lodging: Intermediate Fruiting Branch: Normal Growth: IntermediateLeaf Color: Light Green Boll Shape: Length > Width Boll Breadth:Broadest at middle Maturity: Date of 50% open bolls: 119 days Plant: Cmto 1^(st) Fruiting Branch (from cotyledonary 20.8 node): No. of Nodes to1st Fruiting Branch  5.4 (excluding cotyledonary node): Mature PlantHeight (from cotyledonary 108 cm node to terminal) Leaf (Upper most,fully expanded leaf): Type Normal Pubescence Sparse Nectaries PresentStem Pubescence: Intermediate Glands: Leaf Normal Stem Normal Calyx LobeNormal Flower: Petals Cream Pollen Cream Petal Spot: Absent Seed: SeedIndex (g/100 seed, fuzzy basis):  9.2 Lint Index: (g lint/100 seeds): 6.8 Boll: Lint Percent: Picked 42.6 Number of Seeds per Boll: 29.4Grams of Seed Cotton per Boll:  4.9 Number of Locules per Boll: 4-5 BollType: Open Fiber Properties: Length (inches, 2.5% SL):  1.14 Uniformity(%): 84 Strength, T1 (g/tex): 30.7 Elongation, E1 (%): 11.1 Micronaire: 4.6 Nematodes, Insects and Pests: Root-Knot Nematode SusceptibleReniform Nematode Susceptible Diseases: Fusarium wilt Susceptible Bronzewilt Resistant

[0048] This invention is also directed to methods for producing a cottonplant by crossing a first parent cotton plant with a second parentcotton plant, wherein the first or second cotton plant is the cottonplant from the line 99×35. Further, both the first and second parentcotton plants may be the cultivar 99×35 (e.g., self-pollination).Therefore, any methods using the cultivar 99×35 are part of thisinvention: selfing, backcrosses, hybrid breeding, and crosses topopulations. Any plants produced using cultivar 99×35 as a parent arewithin the scope of this invention. As used herein, the term “plant”includes plant cells, plant protoplasts, plant cells of tissue culturefrom which cotton plants can be regenerated, plant calli, plant clumps,and plant cells that are intact in plants or parts of plants, such aspollen, flowers, embryos, ovules, seeds, pods, leaves, stems, roots,anthers and the like. Thus, another aspect of this invention is toprovide for cells which upon growth and differentiation produce acultivar having essentially all of the physiological and morphologicalcharacteristics of 99×35.

[0049] Culture for expressing desired structural genes and culturedcells are known in the art. Also as known in the art, cotton istransformable and regenerable such that whole plants containing andexpressing desired genes under regulatory control may be obtained.General descriptions of plant expression vectors and reporter genes andtransformation protocols can be found in Gruber, et al., “Vectors forPlant Transformation, in Methods in Plant Molecular Biology &Biotechnology” in Glich, et al., (Eds. pp. 89-119, CRC Press, 1993).Moreover GUS expression vectors and GUS gene cassettes are availablefrom Clone Tech Laboratories, Inc., Palo Alto, Calif. while luciferaseexpression vectors and luciferase gene cassettes are available fromPromega Corp. (Madison, Wis.). General methods of culturing planttissues are provided for example by Maki, et al., “Procedures forIntroducing Foreign DNA into Plants” in Methods in Plant MolecularBiology & Biotechnology, Glich, et al., (Eds. pp. 67-88 CRC Press,1993); and by Phillips, et al., “Cell-Tissue Culture and In-VitroManipulation” in Corn & Corn Improvement, 3rd Edition; Sprague, et al.,(Eds. pp. 345-387) American Society of Agronomy Inc., 1988. Methods ofintroducing expression vectors into plant tissue include the directinfection or co-cultivation of plant cells with Agrobacteriumtumefaciens, Horsch et al., Science, 227:1229 (1985). Descriptions ofAgrobacterium vectors systems and methods for Agrobacterium-mediatedgene transfer provided by Gruber, et al., supra.

[0050] Useful methods include, but are not limited, to expressionvectors introduced into plant tissues using a direct gene transfermethod such as microprojectile-mediated delivery, DNA injection,electroporation and the like. More preferably, expression vectors areintroduced into plant tissues using the microprojectile media deliverywith the biolistic device Agrobacterium-medicated transformation.Transformant plants obtained with the protoplasm of the invention areintended to be within the scope of this invention.

[0051] The present invention contemplates a cotton plant regeneratedfrom a tissue culture of a variety (e.g., 99×35) or hybrid plant of thepresent invention. As is well known in the art, tissue culture of cottoncan be used for the in vitro regeneration of a cotton plant. Tissueculture of various tissues of cotton and regeneration of plantstherefrom is well known and widely published.

[0052] When the term cotton plant is used in the context of the presentinvention, this also includes any single trait conversions of thatvariety. The term single trait converted plant as used herein refers tothose cotton plants which are developed by a plant breeding techniquecalled backcrossing or via genetic engineering wherein essentially allof the desired morphological and physiological characteristics of avariety are recovered in addition to the single trait transferred intothe variety. Backcrossing methods can be used with the present inventionto improve or introduce a characteristic into the variety. The termbackcrossing as used herein refers to the repeated crossing of a hybridprogeny back to the recurrent parent. The parental cotton plant whichcontributes the trait for the desired characteristic is termed thenonrecurrent or donor parent. This terminology refers to the fact thatthe nonrecurrent parent is used one time in the backcross protocol andtherefore does not recur. The parental cotton plant to which the traitor traits from the nonrecurrent parent are transferred is known as therecurrent parent as it is used for several rounds in the backcrossingprotocol (Poehlman & Sleper, 1994; Fehr, 1987). In a typical backcrossprotocol, the original variety of interest (recurrent parent) is crossedto a second variety (nonrecurrent parent) that carries the single geneof interest to be transferred. The resulting progeny from this cross arethen crossed again to the recurrent parent and the process is repeateduntil a cotton plant is obtained wherein essentially all of the desiredmorphological and physiological characteristics of the recurrent parentare recovered in the converted plant, in addition to the singletransferred gene from the nonrecurrent parent.

[0053] The selection of a suitable recurrent parent is an important stepfor a successful backcrossing procedure. The goal of a backcrossprotocol is to alter or substitute a single trait or characteristic inthe original variety. To accomplish this, a gene or genes of therecurrent variety are modified or substituted with the desired gene(s)from the nonrecurrent parent, while retaining essentially all of therest of the desired genetic, and therefore the desired physiological andmorphological, constitution of the original variety. The choice of theparticular nonrecurrent parent will depend on the purpose of thebackcross. One of the major purposes is to add some commerciallydesirable, agronomically important trait to the plant. The exactbackcrossing protocol will depend on the characteristic or trait beingaltered to determine an appropriate testing protocol. Althoughbackcrossing methods are simplified when the characteristic beingtransferred is a dominant allele, a recessive allele may also betransferred. In this instance it may be necessary to introduce a test ofthe progeny to determine if the desired characteristic has beensuccessfully transferred.

[0054] Many traits have been identified that are not regularly selectedfor in the development of a new variety but that can be improved bybackcrossing techniques. Traits may or may not be transgenic, examplesof these traits include but are not limited to, male sterility,herbicide resistance, resistance for bacterial, fungal, or viraldisease, insect resistance, male fertility, enhanced fiber quality,industrial usage, yield stability and yield enhancement. These traitsare generally inherited through the nucleus.

FURTHER EMBODIMENTS OF THE INVENTION

[0055] With the advent of molecular biological techniques that haveallowed the isolation and characterization of genes that encode specificprotein products, scientists in the field of plant biology developed astrong interest in engineering the genome of plants to contain andexpress foreign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreignadditional and/or modified genes are referred to herein collectively as“transgenes”. Over the last fifteen to twenty years several methods forproducing transgenic plants have been developed, and the presentinvention, in particular embodiments, also relates to transformedversions of the claimed variety or line.

[0056] Plant transformation involves the construction of an expressionvector which will function in plant cells. Such a vector comprises DNAcomprising a gene under control of or operatively linked to a regulatoryelement (for example, a promoter). The expression vector may contain oneor more such operably linked gene/regulatory element combinations. Thevector(s) may be in the form of a plasmid, and can be used alone or incombination with other plasmids, to provide transformed cotton plants,using transformation methods as described below to incorporatetransgenes into the genetic material of the cotton plant(s).

[0057] Expression Vectors for Cotton Transformation: MarkerGenes—Expression vectors include at least one genetic marker, operablylinked to a regulatory element (a promoter, for example) that allowstransformed cells containing the marker to be either recovered bynegative selection, i.e., inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or a herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

[0058] One commonly used selectable marker gene for plant transformationis the neomycin phosphotransferase II (nptII) under the control of plantregulatory signals which confers resistance to kanamycin. Fraley et al.,Proc. Natl. Acad. Sci. U.S.A., 80:4803 (1983). Another commonly usedselectable marker gene is the hygromycin phosphotransferase gene whichconfers resistance to the antibiotic hygromycin. Vanden Elzen et al.,Plant Mol. Biol., 5:299 (1985).

[0059] Additional selectable marker genes of bacterial origin thatconfer resistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase,the bleomycin resistance determinant. Hayford et al., Plant Physiol.86:1216 (1988), Jones et al., Mol. Gen. Genet., 210:86 (1987), Svab etal., Plant Mol. Biol. 14:197 (1990) Hille et al., Plant Mol. Biol. 7:171(1986). Other selectable marker genes confer resistance to herbicidessuch as glyphosate, glufosinate or broxynil. Comai et al., Nature317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2:603-618 (1990) andStalker et al., Science 242:419-423 (1988).

[0060] Other selectable marker genes for plant transformation are not ofbacterial origin. These genes include, for example, mouse dihydrofolatereductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plantacetolactate synthase. Eichholtz et al., Somatic Cell Mol. Genet. 13:67(1987), Shah et al., Science 233:478 (1986), Charest et al., Plant CellRep. 8:643 (1990).

[0061] Another class of marker genes for plant transformation requirescreening of presumptively transformed plant cells rather than directgenetic selection of transformed cells for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningpresumptively transformed cells include β-glucuronidase (GUS,β-galactosidase, luciferase and chloramphenicol, acetyltransferase.Jefferson, R. A., Plant Mol. Biol. Rep. 5:387 (1987), Teeri et al., EMBOJ. 8:343 (1989), Koncz et al., Proc. Natl. Acad. Sci U.S.A. 84:131(1987), DeBlock et al., EMBO J. 3:1681 (1984).

[0062] Recently, in vivo methods for visualizing GUS activity that donot require destruction of plant tissue have been made available.Molecular Probes publication 2908, Imagene Green™, p. 1-4 (1993) andNaleway et al., J. Cell Biol. 115:151a (1991). However, these in vivomethods for visualizing GUS activity have not proven useful for recoveryof transformed cells because of low sensitivity, high fluorescentbackgrounds and limitations associated with the use of luciferase genesas selectable markers.

[0063] More recently, a gene encoding Green Fluorescent Protein (GFP)has been utilized as a marker for gene expression in prokaryotic andeukaryotic cells. Chalfie et al., Science 263:802 (1994). GFP andmutants of GFP may be used as screenable markers.

[0064] Promoters—Genes included in expression vectors must be driven bya nucleotide sequence comprising a regulatory element, for example, apromoter. Several types of promoters are now well known in thetransformation arts, as are other regulatory elements that can be usedalone or in combination with promoters.

[0065] As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred”.Promoters which intitiate transcription only in certain tissue arereferred to as “tissue-specific”. A “cell type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter which is active under mostenvironmental conditions.

[0066] A. Inducible Promoters—An inducible promoter is operably linkedto a gene for expression in cotton. Optionally, the inducible promoteris operably linked to a nucleotide sequence encoding a signal sequencewhich is operably linked to a gene for expression in cotton. With aninducible promoter the rate of transcription increases in response to aninducing agent.

[0067] Any inducible promoter can be used in the instant invention. SeeWard et al., Plant Mol. Biol. 22:361-366 (1993). Exemplary induciblepromoters include, but are not limited to, that from the ACEI systemwhich responds to copper (Mett et al., PNAS 90:4567-4571 (1993)); In2gene from maize which responds to benzenesulfonamide herbicide safeners(Hershey et al., Mol. Gen Genetics 227:229-237 (1991) and Gatz et al.,Mol. Gen. Genetics 243:32-38 (1994)) or Tet repressor from Tn10 (Gatz etal., Mol. Gen. Genetics 227:229-237 (1991). A particularly preferredinducible promoter is a promoter that responds to an inducing agent towhich plants do not normally respond. An exemplary inducible promoter isthe inducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone. Schena etal., Proc. Natl. Acad. Sci. U.S.A. 88:0421 (1991).

[0068] B. Constitutive Promoters—A constitutive promoter is operablylinked to a gene for expression in cotton or the constitutive promoteris operably linked to a nucleotide sequence encoding a signal sequencewhich is operably linked to a gene for expression in cotton.

[0069] Many different constitutive promoters can be utilized in theinstant invention. Exemplary constitutive promoters include, but are notlimited to, the promoters from plant viruses such as the 35S promoterfrom CaMV (Odell et al., Nature 313:810-812 (1985) and the promotersfrom such genes as rice actin (McElroy et al., Plant Cell 2:163-171(1990)); ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632(1989) and Christensen et al., Plant Mol. Biol. 18:675-689 (1992)); pEMU(Last et al., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten etal., EMBO J. 3:2723-2730 (1984)) and maize H3 histone (Lepetit et al.,Mol. Gen. Genetics 231:276-285 (1992) and Atanassova et al., PlantJournal 2 (3): 291-300 (1992)).

[0070] The ALS promoter, Xba1/Ncol fragment 5′ to the Brassica napusALS3 structural gene (or a nucleotide sequence similarity to saidXba1/Ncol fragment), represents a particularly useful constitutivepromoter. See PCT application WO 96/30530.

[0071] C. Tissue-specific or Tissue-preferred Promoters—Atissue-specific promoter is operably linked to a gene for expression incotton. Optionally, the tissue-specific promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in cotton. Plants transformed with a gene ofinterest operably linked to a tissue-specific promoter produce theprotein product of the transgene exclusively, or preferentially, in aspecific tissue.

[0072] Any tissue-specific or tissue-preferred promoter can be utilizedin the instant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferredpromoter—such as that from the phaseolin gene (Murai et al., Science23:476-482 (1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci.U.S.A. 82:3320-3324 (1985)); a leaf-specific and light-induced promotersuch as that from cab or rubisco (Simpson et al., EMBO J.4(11):2723-2729 (1985) and Timko et al., Nature 318:579-582 (1985)); ananther-specific promoter such as that from LAT52 (Twell et al., Mol.Gen. Genetics 217:240-245 (1989)); a pollen-specific promoter such asthat from Zm13 (Guerrero et al., Mol. Gen. Genetics 244:161-168 (1993))or a microspore-preferred promoter such as that from apg (Twell et al.,Sex. Plant Reprod. 6:217-224 (1993). Signal Sequences for TargetingProteins to Subcellular Compartments

[0073] Transport of protein produced by transgenes to a subcellularcompartment such as the chloroplast, vacuole, peroxisome, glyoxysome,cell wall or mitochondrion or for secretion into the apoplast, isaccomplished by means of operably linking the nucleotide sequenceencoding a signal sequence to the 5′ and/or 3′ region of a gene encodingthe protein of interest. Targeting sequences at the 5′ and/or 3′ end ofthe structural gene may determine, during protein synthesis andprocessing, where the encoded protein is ultimately compartmentalized.

[0074] The presence of a signal sequence directs a polypeptide to eitheran intracellular organelle or subcellular compartment or for secretionto the apoplast. Many signal sequences are known in the art. See, forexample Becker et al., Plant Mol. Biol. 20:49 (1992), Close, P. S.,Master's Thesis, Iowa State University (1993), Knox, C., et al.,“Structure and Organization of Two Divergent Alpha-Amylase Genes fromBarley”, Plant Mol. Biol. 9:3-17 (1987), Lerneret al., Plant Physiol.91:124-129 (1989), Fontes et al., Plant Cell 3:483496 (1991), Matsuokaet al., Proc. Natl. Acad. Sci. 88:834 (1991), Gould et al., J. Cell.Biol. 108:1657 (1989), Creissen et al., Plant J. 2:129 (1991), Kalderon,et al., A short amino acid sequence able to specify nuclear location,Cell 39:499-509 (1984), Steifel, et al., Expression of a maize cell wallhydroxyproline-rich glycoprotein gene in early leaf and root vasculardifferentiation, Plant Cell 2:785-793 (1990).

[0075] Foreign Protein Genes and Agronomic Genes—With transgenic plantsaccording to the present invention, a foreign protein can be produced incommercial quantities. Thus, techniques for the selection andpropagation of transformed plants, which are well understood in the art,yield a plurality of transgenic plants which are harvested in aconventional manner, and a foreign protein then can be extracted from atissue of interest or from total biomass. Protein extraction from plantbiomass can be accomplished by known methods which are discussed, forexample, by Heney and Orr, Anal. Biochem. 114:92-6 (1981).

[0076] According to a preferred embodiment, the transgenic plantprovided for commercial production of foreign protein is a cotton plant.In another preferred embodiment, the biomass of interest is seed. Forthe relatively small number of transgenic plants that show higher levelsof expression, a genetic map can be generated, primarily viaconventional RFLP, PCR and SSR analysis, which identifies theapproximate chromosomal location of the integrated DNA molecule. Forexemplary methodologies in this regard, see Glick and Thompson, Methodsin Plant Molecular Biology and Biotechnology CRC Press, Boca Raton269:284 (1993). Map information concerning chromosomal location isuseful for proprietary protection of a subject transgenic plant. Ifunauthorized propagation is undertaken and crosses made with othergermplasm, the map of the integration region can be compared to similarmaps for suspect plants, to determine if the latter have a commonparentage with the subject plant. Map comparisons would involvehybridizations, RFLP, PCR, SSR and sequencing, all of which areconventional techniques.

[0077] Likewise, by means of the present invention, agronomic genes canbe expressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

[0078] 1. Genes That Confer Resistance to Pests or Disease and ThatEncode:

[0079] A. Plant disease resistance genes. Plant defenses are oftenactivated by specific interaction between the product of a diseaseresistance gene (R) in the plant and the product of a correspondingavirulence (Avr) gene in the pathogen. A plant variety can betransformed with cloned resistance gene to engineer plants that areresistant to specific pathogen strains. See, for example Jones et al.,Science 266:789 (1994) (cloning of the tomato Cf-9 gene for resistanceto Cladosporium fulvum); Martin et al., Science 262:1432 (1993) (tomatoPto gene for resistance to Pseudomonas syringae pv. Tomato encodes aprotein kinase); Mindrinos et al., Cell 78:1089 (1994) (Arabidopsis RSP2gene for resistance to Pseudomonas syringae).

[0080] B. A gene conferring resistance to a pest, such as nematodes. Seee.g., PCT Application WO 96/30517; PCT Application WO 93/19181.

[0081] C. A Bacillus thuringiensis protein, a derivative thereof or asynthetic polypeptide modeled thereon. See, for example, Geiser et al.,Gene 48:109 (1986), who disclose the cloning and nucleotide sequence ofa Bt δ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxingenes can be purchased from American Type Culture Collection, Manassas,Va., for example, under ATCC Accession Nos. 40098, 67136, 31995 and31998.

[0082] D. A lectin. See, for example, the disclosure by Van Damme etal., Plant Molec. Biol. 24:25 (1994), who disclose the nucleotidesequences of several Clivia miniata mannose-binding lectin genes.

[0083] E. A vitamin-binding protein such as avidin. See PCT applicationUS93/06487, the contents of which are hereby incorporated by reference.The application teaches the use of avidin and avidin homologues aslarvicides against insect pests.

[0084] F. An enzyme inhibitor, for example, a protease or proteinaseinhibitor or an amylase inhibitor. See, for example, Abe et al., J.Biol. Chem. 262:16793 (1987) (nucleotide sequence of rice cysteineproteinase inhibitor), Huub et al., Plant Molec. Biol. 21:985 (1993)(nucleotide sequence of cDNA encoding tobacco proteinase inhibitor I),Sumitani et al., Biosci. Biotech. Biochem. 57:1243 (1993) (nucleotidesequence of Streptomyces nitrosporeus α-amylase inhibitor) and U.S. Pat.No. 5,494,813 (Hepher and Atkinson, issued Feb. 27, 1996).

[0085] G. An insect-specific hormone or pheromone such as an ecdysteroidand juvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

[0086] H. An insect-specific peptide or neuropeptide which, uponexpression, disrupts the physiology of the affected pest. For example,see the disclosures of Regan, J. Biol. Chem. 269:9 (1994) (expressioncloning yields DNA coding for insect diuretic hormone receptor), andPratt et al., Biochem. Biophys. Res. Comm. 163:1243 (1989) (anallostatin is identified in Diploptera puntata). See also U.S. Pat. No.5,266,317 to Tomalski et al., who disclose genes encodinginsect-specific, paralytic neurotoxins.

[0087] I. An insect-specific venom produced in nature by a snake, awasp, etc. For example, see Pang et al., Gene 116:165 (1992), fordisclosure of heterologous expression in plants of a gene coding for ascorpion insectotoxic peptide.

[0088] J. An enzyme responsible for a hyperaccumulation of a monterpene,a sesquiterpene, a steroid, hydroxamic acid, a phenylpropanoidderivative or another non-protein molecule with insecticidal activity.

[0089] K. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637and 67152. Seealso Krameretal., InsectBiochem. Molec. Biol. 23:691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hookworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21:673 (1993), who provide the nucleotide sequence ofthe parsley ubi4-2 polyubiquitin gene.

[0090] L. A molecule that stimulates signal transduction. For example,see the disclosure by Botella et al., Plant Molec. Biol. 24:757 (1994),of nucleotide sequences for mung bean calmodulin cDNA clones, and Griesset al., Plant Physiol. 104:1467 (1994), who provide the nucleotidesequence of a maize calmodulin cDNA clone.

[0091] M. A hydrophobic moment peptide. See PCT application WO 95/16776(disclosure of peptide derivatives of Tachyplesin which inhibit fungalplant pathogens) and PCT application WO 95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance), the respectivecontents of which are hereby incorporated by reference.

[0092] N. A membrane permease, a channel former or a channel blocker.For example, see the disclosure of Jaynes et al., Plant Sci 89:43(1993), of heterologous expression of a cecropin-β, lytic peptide analogto render transgenic tobacco plants resistant to Pseudomonassolanacearum.

[0093] O. A viral-invasive protein or a complex toxin derived therefrom.For example, the accumulation of viral coat proteins in transformedplant cells imparts resistance to viral infection and/or diseasedevelopment effected by the virus from which the coat protein gene isderived, as well as by related viruses. See Beachy et al., Ann. rev.Phytopathol. 28:451 (1990). Coat protein-mediated resistance has beenconferred upon transformed plants against alfalfa mosaic virus, cucumbermosaic virus, tobacco streak virus, potato virus X, potato virus Y,tobacco etch virus, tobacco rattle virus and tobacco mosaic virus. Id.

[0094] P. An insect-specific antibody or an immunotoxin derivedtherefrom. Thus, an antibody targeted to a critical metabolic functionin the insect gut would inactivate an affected enzyme, killing theinsect. Cf. Taylor et al., Abstract #497, Seventh Int'l Symposium onMolecular Plant-Microbe Interactions (Edinburgh, Scotland) (1994)(enzymatic inactivation in transgenic tobacco via production ofsingle-chain antibody fragments).

[0095] Q. A virus-specific antibody. See, for example, Tavladoraki etal., Nature 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

[0096] R. A developmental-arrestive protein produced in nature by apathogen or a parasite. Thus, fungal endo α-1,4-D-polygalacturonasesfacilitate fungal colonization and plant nutrient release bysolubilizing plant cell wall homo-α-1,4-D-galacturonase. See Lamb etal., Bio/Technology 10:1436 (1992). The cloning and characterization ofa gene which encodes a bean endopolygalacturonase-inhibiting protein isdescribed by Toubart et al., Plant J. 2:367 (1992).

[0097] S. A development-arrestive protein produced in nature by a plant.For example, Logemann et al., Bio/Technology 10:305 (1992), have shownthat transgenic plants expressing the barley ribosome-inactivting genehave an increased resistance to fungal disease.

[0098] 2. Genes That Confer Resistance to a Herbicide:

[0099] A. A herbicide that inhibits the growing point or meristem, suchas an imidazolinone or a sulfonylurea. Exemplary genes in this categorycode for mutant ALS and AHAS enzyme as described, for example, by Lee etal., EMBO J. 7:1241 (1988), and Miki et al., Theor. Appl. Genet. 80:449(1990), respectively.

[0100] B. Glyphosate (resistance impaired by mutant5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase, PAT and Streptomyces hygroscopicusphosphinothricin-acetyl transferase, bar, genes), and pyridinoxy orphenoxy proprionic acids and cyclohexones (ACCase inhibitor-encodinggenes). See, for example, U.S. Pat. No. 4,940,835 to Shah, et al., whichdiscloses the nucleotide sequence of a form of EPSP which can conferglyphosate resistance. A DNA molecule encoding a mutant aroA gene can beobtained under ATCC accession number 39256, and the nucleotide sequenceof the mutant gene is disclosed in U.S. Pat. No. 4,769,061 to Comai.European patent application No. 0 333 033 to Kumada et al., and U.S.Pat. No. 4,975,374 to Goodman et al., disclose nucleotide sequences ofglutamine synthetase genes which confer resistance to herbicides such asL-phosphinothricin. The nucleotide sequence of aphosphinothricin-acetyl-transferase gene is provided in Europeanapplication No. 0 242 246 to Leemans et al., DeGreef et al.,Bio/Technology 7:61 (1989), describe the production of transgenic plantsthat express chimeric bar genes coding for phosphinothricin acetyltransferase activity. Exemplary of genes conferring resistance tophenoxy proprionic acids and cyclohexones, such as sethoxydim andhaloxyfop are the Acc1-S1, Acc1-S2 and Acc1-S3 genes described byMarshall et al., Theor. Appl. Genet. 83:435 (1992).

[0101] C. A herbicide that inhibits photosynthesis, such as a triazine(psbA and gs+genes) and a benzonitrile (nitrilase gene). Przibila etal., Plant Cell 3:169 (1991), describe the transformation ofChlamydomonas with plasmids encoding mutant psbA genes. Nucleotidesequences for nitrilase genes are disclosed in U.S. Pat. No. 4,810,648to Stalker, and DNA molecules containing these genes are available underATCC Accession Nos. 53435, 67441, and 67442. Cloning and expression ofDNA coding for a glutathione S-transferase is described by Hayes et al.,Biochem. J. 285:173 (1992).

[0102] 3. Genes That Confer or Contribute to a Value-Added Trait, Suchas:

[0103] A. Modified fatty acid metabolism, for example, by transforming aplant with an antisense gene of stearoyl-ACP desaturase to increasestearic acid content of the plant. See Knultzon et al., Proc. Natl.Acad. Sci. U.S.A. 89:2624 (1992).

[0104] B. Decreased phytate content—1) Introduction of aphytase-encoding gene would enhance breakdown of phytate, adding morefree phosphate to the transformed plant. For example, see VanHartingsveldt et al., Gene 127:87 (1993), for a disclosure of thenucleotide sequence of an Aspergillus niger phytase gene. 2) A genecould be introduced that reduced phytate content. In maize, this, forexample, could be accomplished, by cloning and then reintroducing DNAassociated with the single allele which is responsible for maize mutantscharacterized by low levels of phytic acid. See Raboy et al., Maydica35:383 (1990).

[0105] C. Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza et al., J. Bacteol. 170:810(1988) (nucleotide sequence of Streptococcus mutantsfructosyltransferase gene), Steinmetz et al., Mol. Gen. Genet. 20:220(1985) (nucleotide sequence of Bacillus subtilis levansucrase gene), Penet al., Bio/Technology 10:292 (1992) (production of transgenic plantsthat express Bacillus lichenifonnis α-amylase), Elliot et al., PlantMolec. Biol. 21:515 (1993) (nucleotide sequences of tomato invertasegenes), SØgaard et al., J. Biol. Chem. 268:22480 (1993) (site-directedmutagenesis of barley α-amylase gene), and Fisher et al., Plant Physiol.102:1045 (1993) (maize endosperm starch branching enzyme II).

[0106] Methods for Cotton Transformation—Numerous methods for planttransformation have been developed, including biological and physical,plant transformation protocols. See, for example, Miki et al.,“Procedures for Introducing Foreign DNA into Plants” in Methods in PlantMolecular Biology and Biotechnology, Glick B. R. and Thompson, J. E.Eds. (CRC Press, Inc., Boca Raton, 1993) pages 67-88. In addition,expression vectors and in vitro culture methods for plant cell or tissuetransformation and regeneration of plants are available. See, forexample, Gruber et al., “Vectors for Plant Transformation” in Methods inPlant Molecular Biology and Biotechnology, Glick B. R. and Thompson, J.E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages 89-119.

[0107] A. Agrobacterium-mediated Transformation—One method forintroducing an expression vector into plants is based on the naturaltransformation system of Agrobacterium. See, forexample, Horsch et al.,Science 227:1229 (1985). A. tumefaciens and A. rhizogenes are plantpathogenic soil bacteria which genetically transform plant cells. The Tiand Ri plasmids of A. tumefaciens and A. rhizogenes, respectively, carrygenes responsible for genetic transformation of the plant. See, forexample, Kado, C. I., Crit. Rev. Plant Sci. 10:1 (1991). Descriptions ofAgrobacterium vector systems and methods for Agrobacterium-mediated genetransfer are provided by Gruber et al., supra, Miki et al., supra, andMoloney et al., Plant Cell Reports 8:238 (1989). See also, U.S. Pat. No.5,563,055 (Townsend and Thomas), issued Oct. 8, 1996.

[0108] B. Direct Gene Transfer—Several methods of plant transformation,collectively referred to as direct gene transfer, have been developed asan alternative to Agrobacterium-mediated transformation. A generallyapplicable method of plant transformation is microprojectile-mediatedtransformation wherein DNA is carried on the surface of microprojectilesmeasuring 1 to 4 μm. The expression vector is introduced into planttissues with a biolistic device that accelerates the microprojectiles tospeeds of 300 to 600 m/s which is sufficient to penetrate plant cellwalls and membranes. Sanford et al., Part. Sci. Technol. 5:27 (1987),Sanford, J. C., Trends Biotech. 6:299 (1988), Klein et al.,Bio/Technology 6:559-563 (1988), Sanford, J.C., Physiol Plant 7:206(1990), Klein et al., Biotechnology 10:268 (1992). See also U.S. Pat.No. 5,015,580 (Christou, et al.), issued May 14, 1991; U.S. Pat. No.5,322,783 (Tomes, et al.), issued Jun. 21, 1994.

[0109] Another method for physical delivery of DNA to plants issonication of target cells. Zhang et al., Bio/Technology 9:996 (1991).Alternatively, liposome or spheroplast fusion have been used tointroduce expression vectors into plants. Deshayes et al., EMBO J.,4:2731 (1985), Christou et al., Proc Natl. Acad. Sci. U.S.A. 84:3962(1987). Direct uptake of DNA into protoplasts using CaCl₂ precipitation,polyvinyl alcohol or poly-L-omithine have also been reported. Hain etal., Mol. Gen. Genet. 199:161 (1985) and Draper et al., Plant CellPhysiol. 23:451 (1982). Electroporation of protoplasts and whole cellsand tissues have also been described. Donn et al., In Abstracts of VIIthInternational Congress on Plant Cell and Tissue Culture IAPTC, A2-38, p53 (1990); D'Halluin et al., Plant Cell 4:1495-1505 (1992) and Spenceret al., Plant Mol. Biol. 24:51-61 (1994).

[0110] Following transformation of cotton target tissues, expression ofthe above-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

[0111] The foregoing methods for transformation would typically be usedfor producing a transgenic variety. The transgenic variety could then becrossed, with another (non-transformed or transformed) variety, in orderto produce a new transgenic variety. Alternatively, a genetic traitwhich has been engineered into a particular cotton line using theforegoing transformation techniques could be moved into another lineusing traditional backcrossing techniques that are well known in theplant breeding arts. For example, a backcrossing approach could be usedto move an engineered trait from a public, non-elite variety into anelite variety, or from a variety containing a foreign gene in its genomeinto a variety or varieties which do not contain that gene. As usedherein, “crossing” can refer to a simple X by Y cross, or the process ofbackcrossing, depending on the context.

[0112] Tissue Culture of Cotton—When the term “cotton plant” is used inthe context of the present invention, this also includes any single geneconversions of that variety. The term “single gene converted plant” asused herein refers to those cotton plants which are developed by a plantbreeding technique called backcrossing wherein essentially all of thedesired morphological and physiological characteristics of a variety arerecovered in addition to the single gene transferred into the varietyvia the backcrossing technique. Backcrossing methods can be used withthe present invention to improve or introduce a characteristic into thevariety. The term “backcrossing” as used herein refers to the repeatedcrossing of a hybrid progeny back to the recurrent parent, i.e.,backcrossing 1, 2, 3, 4, 5, 6, 7 or more times to the recurrent parent.The parental cotton plant which contributes the gene for the desiredcharacteristic is termed the “nonrecurrent” or “donor parent”. Thisterminology refers to the fact that the nonrecurrent parent is used onetime in the backcross protocol and therefore does not recur. Theparental cotton plant to which the gene or genes from the nonrecurrentparent are transferred is known as the recurrent parent as it is usedfor several rounds in the backcrossing protocol (Poehiman & Sleper,1994; Fehr, 1987). In a typical backcross protocol, the original varietyof interest (recurrent parent) is crossed to a second variety(nonrecurrent parent) that carries the single gene of interest to betransferred. The resulting progeny from this cross are then crossedagain to the recurrent parent and the process is repeated until a cottonplant is obtained wherein essentially all of the desired morphologicaland physiological characteristics of the recurrent parent are recoveredin the converted plant, in addition to the single transferred gene fromthe nonrecurrent parent.

[0113] The selection of a suitable recurrent parent is an important stepfor a successful backcrossing procedure. The goal of a backcrossprotocol is to alter or substitute a single trait or characteristic inthe original variety. To accomplish this, a single gene of the recurrentvariety is modified or substituted with the desired gene from thenonrecurrent parent, while retaining essentially all of the rest of thedesired genetic, and therefore the desired physiological andmorphological, constitution of the original variety. The choice of theparticular nonrecurrent parent will depend on the purpose of thebackcross. One of the major purposes is to add some commerciallydesirable, agronomically important trait to the plant. The exactbackcrossing protocol will depend on the characteristic or trait beingaltered to determine an appropriate testing protocol. Althoughbackcrossing methods are simplified when the characteristic beingtransferred is a dominant allele, a recessive allele may also betransferred. In this instance it may be necessary to introduce a test ofthe progeny to determine if the desired characteristic has beensuccessfully transferred.

[0114] Many single gene traits have been identified that are notregularly selected for in the development of a new variety but that canbe improved by backcrossing techniques. Single gene traits may or maynot be transgenic, examples of these traits include but are not limitedto, male sterility, waxy starch, herbicide resistance, resistance forbacterial, fungal, or viral disease, insect resistance, male fertility,enhanced nutritional quality, industrial usage, yield stability andyield enhancement. These genes are generally inherited through thenucleus. Several of these single gene traits are described in U.S. Pat.Nos. 5,959,185, 5,973,234 and 5,977,445, the disclosures of which arespecifically hereby incorporated by reference.

[0115] Further reproduction of the variety can occur by tissue cultureand regeneration. Tissue culture of various tissues of cotton andregeneration of plants therefrom is well known and widely published. Forexample, reference may be had to Komatsuda, T. et al., “Genotype XSucrose Interactions for Somatic Embryogenesis in Soybean,” Crop Sci.31:333-337 (1991); Stephens, P. A., et al., “Agronomic Evaluation ofTissue-Culture-Derived Soybean Plants,” Theor. Appl. Genet. (1991)82:633-635; Komatsuda, T. et al., “Maturation and Germination of SomaticEmbryos as Affected by Sucrose and Plant Growth Regulators in SoybeansGlycine gracilis Skvortz and Glycine max (L.) Merr.” Plant Cell, Tissueand Organ Culture, 28:103-113 (1992); Dhir, S. et al., “Regeneration ofFertile Plants from Protoplasts of Soybean (Glycine max L. Merr.);Genotypic Differences in Culture Response,” Plant Cell Reports (1992)11:285-289; Pandey, P. et al., “Plant Regeneration from Leaf andHypocotyl Explants of Glycine-wightii (W. and A.) VERDC. var.longicauda,” Japan J. Breed. 42:1-5 (1992); and Shetty, K., et al.,“Stimulation of In Vitro Shoot Organogenesis in Glycine max (Merrill.)by Allantoin and Amides,” Plant Science 81:245-251 (1992); as well asU.S. Pat. No. 5,024,944 issued Jun. 18, 1991 to Collins et al., and U.S.Pat. No. 5,008,200 issued Apr. 16, 1991 to Ranch et al., the disclosuresof which are hereby incorporated herein in their entirety by reference.Thus, another aspect of this invention is to provide cells which upongrowth and differentiation produce cotton plants having thephysiological and morphological characteristics of cotton variety 99×35.

[0116] As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, plant clumps, and plantcells that can generate tissue culture that are intact in plants orparts of plants, such as embryos, pollen, flowers, seeds, pods, leaves,stems, roots, root tips, anthers, and the like. Means for preparing andmaintaining plant tissue culture are well known in the art. By way ofexample, a tissue culture comprising organs has been used to produceregenerated plants. U.S. Pat. Nos. 5,959,185, 5,973,234 and 5,977,445,described certain techniques, the disclosures of which are incorporatedherein by reference.

[0117] This invention also is directed to methods for producing a cottonplant by crossing a first parent cotton plant with a second parentcotton plant wherein the first or second parent cotton plant is a cottonplant of the variety 99×35. Further, both first and second parent cottonplants can come from the cotton variety 99×35. Thus, any such methodsusing the cotton variety 99×35 are part of this invention: selfing,backcrosses, hybrid production, crosses to populations, and the like.All plants produced using cotton variety 99×35 as a parent are withinthe scope of this invention, including those developed from varietiesderived from cotton variety 99×35. Advantageously, the cotton varietycould be used in crosses with other, different, cotton plants to producefirst generation (F₁) cotton hybrid seeds and plants with superiorcharacteristics. The variety of the invention can also be used fortransformation where exogenous genes are introduced and expressed by thevariety of the invention. Genetic variants created either throughtraditional breeding methods using variety 99×35 or throughtransformation of 99×35 by any of a number of protocols known to thoseof skill in the art are intended to be within the scope of thisinvention.

[0118] As shown in Tables 2 and 3 below, 99×35 is compared to othercotton varieties. Column one shows the varieties, columns 2 and 3 showthe lint yield expressed in kilograms of lint per hectare and lintpercent. Columns 4, 5, 6 and 7 list micronaire (Mic), fiber length(Len), uniformity ratio (UR) and fiber strength (T1) valuesrespectively. Columns 8, 9, 10, 11 and 12 show the fiber elongation(E1), plant height in inches; maturity rating; stringout and falloutratings. TABLE 2 Summary across Southeast, Lower Midsouth and SouthTexas, 1998-2000 Lint Plt Variety Yield Lint % Mic Len UR T1 E1 Hgt Mat.Stringout Fallout 99X35 1139 40.4 4.5 1.08 83 27.7 11.0 40 1.8 2.7 2.5ST 474 1058 38.2 4.6 1.08 83 28.1 10.9 41 2.4 3.1 1.9 Locs 32 33 33 3333 33 17 10 6 17 10

[0119] TABLE 3 Summary across Southeast, Lower Midsouth and South Texas,1999-2000 Lint Plt Variety Yield Lint % Mic Len UR T1 E1 Hgt Mat.Stringout Fallout 99X35 1074 40.5 4.5 1.08 83 27.6 11.1 40 1.9 2.7 2.5ST 474 1011 38.5 4.5 1.08 83 28.1 11.0 41 2.3 3.0 1.9 SG 747 980 38.04.6 1.09 83 27.8 12.4 39 1.9 3.0 1.8 Locs 22 22 22 22 22 22 15 10 5 15 8

DEPOSIT INFORMATION

[0120] A deposit of the cotton seed of this invention is maintained byDelta and Pine Land Company, 100 Main Street, Scott, Miss. 38772. Accessto this deposit will be available during the pendency of thisapplication to persons determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 CFR 1.14 and 35 USC 122. Uponallowance of any claims in this application, all restrictions on theavailability to the public of the variety will be irrevocably removed byaffording access to a deposit of at least 2,500 seeds of the samevariety with the American Type Culture Collection, Manassas, Virginia orNational Collections of Industrial, Food and Marine Bacteria (NCIMB), 23St Machar Drive, Aberdeen, Scotland, AB24 3RY, United Kingdom.

[0121] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity andunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the invention, as limited only bythe scope of the appended claims.

What is claimed is:
 1. Seed of cotton line designated 99×35,representative seed of said line having been deposited under ATCCAccession No. PTA-______.
 2. A cotton plant, or a part thereof, producedby growing the seed of claim
 1. 3. A tissue culture of regenerable cellsproduced from the plant of claim
 2. 4. Protoplasts produced from thetissue culture of claim
 3. 5. The tissue culture of claim 3, whereincells of the tissue culture are from a tissue selected from the groupconsisting of leaf, pollen, embryo, root, root tip, anther, pistil,flower, seed, boll and stem.
 6. A cotton plant regenerated from thetissue culture of claim 3, said plant having all the morphological andphysiological characteristics of line 99×35, representative seed of saidline having been deposited under ATCC Accession No. PTA-______.
 7. Amethod for producing an F1 hybrid cotton seed, comprising crossing theplant of claim 2 with a different cotton plant and harvesting theresultant F1 hybrid cotton seed.
 8. A hybrid cotton seed produced by themethod of claim
 7. 9. A hybrid cotton plant, or parts thereof, producedby growing said hybrid seed of claim
 8. 10. A method for producing amale sterile cotton plant comprising transforming the cotton plant ofclaim 2 with a nucleic acid molecule that confers male sterility.
 11. Amale sterile cotton plant produced by the method of claim
 10. 12. Amethod of producing an herbicide resistant cotton plant comprisingtransforming the cotton plant of claim 2 with a transgene that confersherbicide resistance.
 13. An herbicide resistant cotton plant producedby the method of claim
 12. 14. The cotton plant of claim 13, wherein thetransgene confers resistance to an herbicide selected from the groupconsisting of: imidazolinone, sulfonylurea, glyphosate, glufosinate,L-phosphinothricin, triazine and benzonitrile.
 15. A method of producingan insect resistant cotton plant comprising transforming the cottonplant of claim 2 with a transgene that confers insect resistance.
 16. Aninsect resistant cotton plant produced by the method of claim
 15. 17.The cotton plant of claim 16, wherein the transgene encodes a Bacillusthuringiensis endotoxin.
 18. A method of producing a disease resistantcotton plant comprising transforming the cotton plant of claim 2 with atransgene that confers disease resistance.
 19. A disease resistantcotton plant produced by the method of claim
 18. 20. A method ofproducing a cotton plant with modified fatty acid metabolism or modifiedcarbohydrate metabolism comprising transforming the cotton plant ofclaim 2 with a transgene encoding a protein selected from the groupconsisting of stearyl-ACP desaturase, fructosyltransferase,levansucrase, alpha-amylase, invertase and starch branching enzyme. 21.A cotton plant produced by the method of claim
 20. 22. A cotton plant,or part thereof, having all the physiological and morphologicalcharacteristics of the line 99×35, representative seed of said linehaving been deposited under ATCC Accession No. PTA-______.
 23. A methodof introducing a desired trait into cotton line 99×35 comprising: (a)crossing 99×35 plants grown from 99×35 seed, representative seed ofwhich has been deposited under ATCC Accession No. PTA-______, withplants of another cotton line that comprise a desired trait to produceF1 progeny plants, wherein the desired trait is selected from the groupconsisting of male sterility, herbicide resistance, insect resistanceand disease resistance; (b) selecting F1 progeny plants that have thedesired trait to produce selected F1 progeny plants; (c) crossing theselected progeny plants with the 99×35 plants to produce backcrossprogeny plants; (d) selecting for backcross progeny plants that have thedesired trait and physiological and morphological characteristics ofcotton line 99×35 listed in Table 1 to produce selected backcrossprogeny plants; and (e) repeating steps (c) and (d) one or more times insuccession to produce selected second or higher backcross progeny plantsthat comprise the desired trait and all of the physiological andmorphological characteristics of cotton line 99×35 listed in Table 1 asdetermined at the 5% significance level when grown in the sameenvironmental conditions.
 24. A plant produced by the method of claim23, wherein the plant has the desired trait and all of the physiologicaland morphological characteristics of cotton line 99×35 listed in Table 1as determined at the 5% significance level when grown in the sameenvironmental conditions.
 25. The plant of claim 24 wherein the desiredtrait is herbicide resistance and the resistance is conferred to anherbicide selected from the group consisting of: imidazolinone,sulfonylurea, glyphosate, glufosinate, L-phosphinothricin, triazine andbenzonitrile.
 26. The plant of claim 24 wherein the desired trait isinsect resistance and the insect resistance is conferred by a transgeneencoding a Bacillus thuringiensis endotoxin.
 27. The plant of claim 24wherein the desired trait is male sterility and the trait is conferredby a cytoplasmic nucleic acid molecule that confers male sterility.